The responses of cells to high levels of sterols has important implications for disease processes and normal physiology. In one setting, the accumulation of free cholesterol (FC) by atheroma macrophages (Mfs) leads to Mf death, which likely promotes plaque instability and acute clinical events, like myocardial infarction. In another setting, intestinal epithelial cells (IECs) are exposed to high levels of potentially toxic dietary and biliary cholesterol and dietary plant sterols, and so mechanisms are necessary to prevent intestinal dysfunction. The PI has recently discovered that a complex signal transduction pathway in the endoplasmic reticulum (ER)--the Unfolded Protein Response (UPR)--plays a critical role in the response of cultured Mrs to FC. The UPR first triggers a set of adaptive response, but then induces apoptosis if the adaptive responses fail. As such, the UPR initially promotes survival in FC-Ioaded Mfs, but then promotes apoptosis after prolonged FC loading. Because a unique branch of the UPR is localized to the gastrointestinal tract, and because a critical enzyme that protects IECs from sterol cytotoxicity is induced by the UPR, the UPR may also play an important role in the protection of IECs from high levels of sterols. In this context, the overall objective of this proposal is to investigate the roles and mechanisms of the UPR in adaptive and pathophysiological processes associated with the accumulation of excess sterols by atheroma Mfs and IECs. This objective will be met by: (a) determining if null mutations in two key UPR genes--one involved in the adaptive response and the other in apoptosis--affect lesional Mf death and plaque instability in the apoE-/- mouse model of atherosclerosis; (b) determining if the UPR is induced in IECs of mice exposed to excess dietary and plant sterols; if proteins known to help protect IECs from sterols are induced by the UPR; and if disruption of the UPR leads to intestinal dysfunction; and (c) investigating how FC induces the UPR by testing the hypothesis that FC enrichment of the ER membrane depletes ER calcium, which is a well-known and potent inducer of the UPR, by inhibiting the sarco/endoplasmic reticulum calcium ATPase or by activating ER calcium channels. The Mf studies will add to our understanding of how plaques become vulnerable and will suggest novel therapeutic targets involved in acute vascular events, and the intestinal studies will reveal a new and important aspect of intestinal-sterol physiology.